Process for producing female-sterile plants

ABSTRACT

The invention relates to processes for producing transgenic female-sterile plants using pistil or stigma tissue-specific promoters. In these plants, the development of particular plant parts can be prevented deliberately.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 9/147,993, filed Mar. 24, 1999 now U.S. Pat. No. 6,759,572 which isthe U.S. National Phase application of International application SerialNo. PCT/EP97/05037, filed Sep. 15, 1997 and claiming priority to Germanapplication Serial No. 196 39 463.5, filed Sep. 26, 1996. Each of theforegoing applications, and each document cited or referenced in each ofthe foregoing applications and during the prosecution of each of theforegoing applications (“application cited documents”), and eachdocument referenced or cited in each of the application cited documents,and each document cited or referenced in this application (“herein citeddocuments”) and each document cited or referenced in each of the hereincited documents, as well as all documents of Applicants duringprosecution of each of the foregoing applications, are all herebyincorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to the use of deacetylase genes for producingtransgenic plants while employing tissue-specific promoters. In theseplants, the development of particular plant parts can be preventeddeliberately.

BACKGROUND OF THE INVENTION

Phosphinothricin (PTC, 2-amino-4-methylphosphinobutyric acid) is aninhibitor of glutamine synthetase (GS). PTC is a “building block” of theantibiotic phosphinothricylalanylalanine. This tripeptide (PTT) isactive against Gram-positive and Gram-negative bacteria and also againstthe fungus Botrytis cinerea. PTT is produced by the Streptomycesviridochromogenes strain Tü494, which is deposited in the DeutscheSammlung für Mikroorganismen (German collection of microorganisms) undernumbers DSM 40736 and DSM 4112 and which is obtainable from this source.It is known from German patent specification 2 717 440 that PTC acts asa total herbicide. The published application (EP-A-0257542)(corresponding U.S. Pat. No. 5,273,894) describes how a phosphinothricinN-acetyltransferase (pat) gene can be used to produceherbicide-resistant plants. The phosphinothricin N-acetyltransferasewhich is encoded by the pat gene modifies the PTC which appearsintracellularly and detoxifies the herbicide.

The present invention now describes the use of deacetylase genes (dea),whose expression products are able to deacetylateN-acetylphosphinothricin (N-Ac-PTC) and/or N-Ac-PTT intracellularly, andthereby restore their antibiotic activity, for producing female-sterileplants.

An N-acetylphosphinothricin tripeptide deacetylase gene can be isolatedfrom S. viridochromogenes Tü494. The dea gene is located downstream ofthe pat gene on the already known 4.0 kb BamHI fragment (EP-A-0 257 542)(corresponding U.S. Pat. No. 5,273,894). This gene is located on aBgIII/BamHI fragment and is fixed precisely by the sequence (FIG. 1 andTab. 1). The protein sequence is defined by the DNA sequence.

An ATG codon, which is recognized in bacteria and plants, is used as thetranslation start codon; the Shine-Dalgarno sequence is underlined. Thisgene encodes the last step in the biosynthesis of PTT, i.e. thedeacetylation of inactive N-acetylphosphinothricin tripeptide to givethe active PTT.

It is known that the specificity of many enzymes is not restricted toone substrate. Thus, the phosphinothricin N-acetyltransferase which isencoded by the pat gene is actually used in PTT biosynthesis foracetylating desmethyl-PTC and, because of its lack of specificity, canbe used for detoxifying PTC. By means of overexpressing the dea gene(using suitable promoters or by cloning onto high-copy vectors), aninsufficiently specific N-acetyl-PTT deacetylase can now be employed foractivating N-acetylphosphinothricin.

Other dea genes can be isolated from E. coli. Thus, it has been foundthat in E. coli, in contrast to other bacteria (e.g. rhizobias andstreptomycetes), no activity can be detected in the so-called pat assay(dissertation of Inge Broer, University of Bielefeld Faculty of Biology,Expression des Phosphinthricin-N-Acetyltransferase-Gens aus Streptomycesviridochromogenes in Nicotiana tabacum (Expression of the Streptomycesviridochromogenes phosphinothricin N-acetyltransferase gene in Nicotianatabacum), pp. 42-43, 1989) after the pat gene has been cloned intosuitable expression vectors (Strauch et al., Gene, 63, 65-74, 1988;Wohlleben et al., Gene, 70, 25-37, 1988). In addition, when present inlow copy number in E. coli, the pat gene is unable to confer resistanceto PTT since the endogenous deacetylase nullifies the effect of thephosphinothricin N-acetyltransferase. Finally, this deacetylase activitycan be demonstrated directly by the efficient inhibition of GS activitywhich occurs after adding N-acetylphosinothricin. The deacetylaseconverts N-Ac-PTC into PTC, which then inhibits the GS in a knownmanner, as can be measured in a γ-glutamyltransferase assay (Bender etal., J. Bacteriol. 129, 1001-1009, 1977). This is due to the possessionby E. coli of an endogenous deacetylase activity.

This activity is apparently not present in the argE mutant which isknown from the literature (Baumberg, Molec. Gen. Genetics 106, 162-173,1970). Other E. coli deacetylase mutants are easy to select: followingclassical (Delić et al., Mut. Res. 9, 167-182, 1970; Drake and Baltz,Ann. Rev. Biochem. 45, 11-38, 1976) or Tn5 mutagenesis (Kleckner, Ann.Rev. Genet. 15, 341-404, 1981), such mutants can be recognized onPTT-supplemented minimal medium by the fact that it is only they whichare able to grow after having been transformed with a pat gene which iscloned into a low copy number vector.

The E. coli deacetylase gene can therefore be isolated by usingconventional methods (Maniatis et al., Molecular Cloning: a LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982)to construct a gene library in, for example, the E. coli argE mutant orin a freshly isolated mutant.

Methods for isolating other deacetylase genes can be inferred from thatwhich is described above: e.g. isolating new organisms which arePTT-sensitive despite the presence of a pat gene on a low copy numbervector, and subsequently isolating a deacetylase gene.

OBJECTS AND SUMMARY OF THE INVENTION

In a further aspect of the invention, pat and dea genes can be employedtogether with tissue-specific promoters for deliberately preventing thedevelopment of particular plant tissues. An example of a specialapplication is that of producing female-sterile plants.

DETAILED DESCRIPTION

In plant breeding, the production of hybrid seed depends on avoidingself-fertilization of the parent plant with a high degree of certainty.Male-sterile mutants, which are employed in breeding, occur naturally inmany plant species. The molecular mechanism of cytoplasmic malesterility (cms) has not so far been completely clarified. In addition,many crop varieties, such as Beta vulgaris, do not have any cms variant.It is therefore of great interest to agriculture to use the geneticengineering route to generate defined sterile mutants of all theimportant crop varieties. The company PGS/Belgium has presented such amethod in patent application PCT/EP 89/00495. This method is based ondestroying the tissue (tapetum) surrounding the pollen parent cells. Forthis purpose, an RNAse gene is fused to a tapetum-specific promoter(Mariani et al., Nature 347, 737-741, 1990). The exclusive expression ofthe gene in the tapetum cells ensures that the tissue is destroyedselectively and thereby prevents the mature pollen from being formed.According to this patent, a plant which carries this gene is only ableto form seeds following allogamy.

An important disadvantage of this system is the fact that the progeny ofthis plant are likewise male-sterile and are therefore unable to formany seeds in the field, where they have to rely on self-fertilization.Success in forming seeds is only achieved if the male partner of thecross carries a gene which is able to neutralize the effect of the RNAsein the progeny. According to the abovementioned laid-open patentapplication, this is supposed to be effected by the barstar gene. Infact, it is only genetically modified, i.e. transgenic partners whichcan be used in the cross in this context.

Processes for producing female-sterile plants (fs plants), whichprocesses allow transgenic parent plants to be crossed with any partnersof the same species, are presented below. This is achieved by thecombination of a dea gene under the control of a promoter which isselectively active in the female organs, where appropriate incombination with a constitutively expressed pat gene. The glutaminesynthetase in the cells is specifically inhibited, and these cells arecaused to die, by applying PTC and/or PTT. An even simpler systemcomprises producing transgenic plants which only contain one singleforeign gene, namely a dea gene under the control of a tissue-specific,in this case female-specific promoter, and then applying N-Ac-PTC and/orN-Ac-PTT to the plant.

To generalize, the invention consequently comprises tissue-specificinhibition with the aid of a deacetylase gene.

1) Plants which are resistant to PTT and/or PTC as a result of Patactivity (e.g. produced as described in EP 0257542 (corresponding U.S.Pat. No. 5,273,894) or EP 0 242 236) are transformed with a deacetylasegene under the control of the promoter which exhibits tissue-specificactivity in plants. Following application of PTT or PTC, expression ofthe deacetylase gene leads to the activity of phosphinothricinN-acetyltransferase being neutralized in the corresponding tissues.These tissues are then killed selectively whereas the remainder of theplant is resistant.

This system can be simplified by using N-acetylphosphinothricin orN-acetylphosphinothricin-tripeptide.-While neither substance isherbicidally active, they are both taken up by plants and transportedand not degraded immediately. No deacetylation activity forN-acetylphosphinothricin and N-acetylphosphinothricin-tripeptide has sofar been demonstrated in plants. In this way, the above-described 2-genesystem can be reduced to a 1-gene system and thereby cruciallysimplified, as explained in more detail below: any plants can betransformed with a Streptomycetes-derived deacetylase gene under thecontrol of a tissue-specific promoter. Following application ofN-acetylphosphinothricin or N-acetylphosphinothricin-tripeptide, thetissue-specific expression leads to the immediate death of thecorresponding tissue.

All the described promoters which have been demonstrated to elicitselective expression in particular tissues, preferably the femaleorgans, can be used as tissue-specific promoters. In this connection,the term female organs encompasses the gametophyte and the tissue whichsurrounds or adjoins it, for example gynoecium (carpels), ovules,placenta, pistil (ovary, style and stigma).

Thus, Robert et al., for example, describe rape-derived stigma-specificpromoters (Robert et al., 1994). Pistil-specific promotes have also beendescribed (Sato et al., 1991; Dzelzkalns et al., 1993, WO 94/25613)(corresponding U.S. Pat. No. 5,859,328).

However, promoters which, while not being specifically active in thefemale organs, are nevertheless expressed in a tissue which is essentialfor the development of the functional flower, embryo and seed, are alsosuitable for use in the process according to the invention.

All newly isolated promoters having similar properties are, of course,also suitable. Apart from tissue-specific promoters, those promoterswhich are subject to another type of regulation (e.g. temporal,stress-determined or environment-dependent) and which behaves in atissue-specific manner can also be employed.

These processes furthermore make it possible to analyze thedifferentiation of cell regulation and to produce plants in which thedevelopment of particular plant parts has been deliberately prevented.The process can preferably be employed for producing female-sterileplants.

EXAMPLES Example 1 Fusing the Deacetylase-Encoding Region to EukaryoticTranscription Signals

The plasmid pPRI (see EP-0 257 542) (corresponding U.S. Pat. No.5,273,894) was isolated from an E. coli strain and cleaved with BamHIand BgIII. The digested DNA was fractionated in an agarose gel, and an0.9 kb fragment was isolated from the gel. The vector pROKI (Baulcombeet al., Nature 321, 446-449, 1986) was likewise restricted with BamHI.The two mixtures were combined and ligated. The ligation mixture wastransformed according to E. coli S17.1 (Simon et al., Bio/Technology 1,784-791, 1983). Colonies which grew on kanamycin-containing media weretransferred to nitrocellulose filters and lysed after being incubated at37° C. for 12 h. The bacterial DNA was fixed to the filter. The 0.9 kbfragment which was isolated from the agarose gel was renderedsingle-stranded by incubation at 100° C. The missing strand was thensynthesized using Klenow polymerase and digoxigenin-labeled nucleotides.The labeled strand was used as the probe for hybridizing with thebacterial DNA which was bound to the filter. Hybridizing clones weredetected by means of an antibody reaction. The DNA of the positiveclones was isolated by means of Qiagen lysis and digested withBamHI/EcoRI and BamHI/HindIII. This restriction enables the orientationof the inserted 0.9 kb fragment to be determined. The plasmid havingorientation I was designated pIB17.1, while that having orientation 11was designated pIB17.2 (see FIG. 2).

Example 2 Detecting the Deacetylation of N-Acetyl-PTC and N-Acetyl-PTTby the Deacetylase Gene

It was possible to demonstrate that the eukaryotic transcription signalscloned in vector pROKI also permit expression in R. meliloti, A.tumefaciens and E. coli.

Plasmids pIB17.1 and pIB17.2 were therefore transferred by means of a 2factor cross into the Rhizobium meliloti strain 2011. By incubating R.meliloti wild-type strains with radioactively labeled N-acetyl-PTC, itwas possible to demonstrate that this strain does not deacetylateN-acetyl-PTC. (After incubating pIB17.1-harboring strains withN-acetyl-PTC and N-acetyl-PTT, the deacetylation can be demonstrated bymeans of thin layer chromatography). It was also possible to demonstratethat R. meliloti reacts very sensitively to PTC and PTT. Thedeacetylation can therefore also be demonstrated by means of theinhibition of the R. meliloti glutamine synthetases which is broughtabout by the liberated PTC.

Example 3 Transferring the Modified Deacetylase Gene Into Nicotianatabacum

The deacetylase gene which was modified in Example 1 was transferredinto A. tumefaciens LBA4404 using a two-factor cross. Nicotiana tabacumleaf disks were incubated with the resulting strains LBA4404/17.1 andLBA4404/17.2 and, after 3 days, transferred to a kanamycin-containingshoot-inducing medium. Southern hybridization can be used to testregenerating kanamycin-resistant shoots for the presence of thedeacetylase gene. Following treatment with N-acetyl-PTC or N-acetyl-PTT,the plants are then killed by the PTC or PTT, respectively, which isliberated.

Example 4 Constructing a Vector for Transiently Expressing the ModifiedDeacetylase Gene in E. coli and Tobacco Protoplasts

The modified deacetylase gene from pIB17.1 and pIB17.2 was excised fromthe plasmids by means of EcoRI/HindIII digestion. The restricted DNA wasfractionated in an agarose gel, and an 0.9 kb fragment was isolated ineach case. The vector pSVB28 (Arnold and Pühler, Gene 70, 171-179, 1988)was likewise digested with EcoRI/HindIII. The two mixtures were combinedand ligated. Following transformation into the β-galactosidase-negativeE. coli strain JM83, all the vector-harboring clones exhibited a bluecoloration whereas clones harboring a vector into which the deacetylasegene was inserted remained white. The DNA was isolated from the cloneswhich had been identified in this way and digested with EcoRI/HindIII.It was possible to identify the clones containing the modifieddeacetylase gene on the basis of the restriction pattern. The vectorswhich were constructed have the designations pIB27.1 and pIB27.2 (seeFIG. 2). They are present in high copy number in E. coli.

Example 5 Transiently Expressing the Modified Deacetylase Gene inTobacco Protoplasts

The plasmid DNA was isolated from the E. coli strains constructed inExample 4. Young tobacco leaves were incubated with digestion enzymesfor 20 h. The protoplasts precipitating from the leaf skeleton werepurified and incubated in a transfer buffer containing polyethyleneglycol (PEG) and the isolated DNA. The protoplasts were subsequentlywashed and resuspended in a culture fluid (K3 medium). After having beenincubated for 3 days under dim illumination, the regeneratingprotoplasts were disrupted and the crude extracts were incubated withradioactively labeled N-acetyl-PTC and N-acetyl-PTT. The deacetylatedPTC or PTT, respectively, can be detected by means of thin layerchromatography.

Example 6 Process for Producing Male-Sterile Crop Plants Using the S.viridochromogenes Deacetylase Gene Under the Control of aTapetum-Specific Promoter

The Streptomyces viridochromogenes deacetylase gene is fused to apistil-specific promoter and introduced into tobacco cells by way ofAgrobacterium-mediated leaf disk transformation. At an arbitrary timebefore flowering, the plants which regenerate from these cells areinjected with N-acetyl-PTC or N-acetyl-PTT. It can be shown thatN-acetyl-PTC is stable in the plant cell and transported into all cells.Neither of the two substances has recognizably negative consequences forthe wild-type plant. As soon as the first pistil cells form, they beginto express the deacetylase gene. The N-acetyl-PTC or N-acetyl-PTT whichis stored in the cell is deacetylated by the enzyme and therebyconverted into its active form. It inhibits the glutamine synthetase ofthe cells and thereby leads to rapid death. Functional embryos or seedscan no longer be formed. Despite this, the development of the maleorgans of reproduction is impaired. In addition, the formation of thedeacetylase is also interrupted. Surrounding cells are not damaged. Ifthe plant is not treated with N-acetyl-PTC or N-acetyl-PTT, it iscompletely fertile. It is not, therefore, necessary to neutralize the fswith a gene of the male partner of the cross. At the same time, theplant contains a precisely defined mutation which is without effect onits vigor and utilizability.

References

-   Dzelzkalns et al., The Plant Cell, Vol. 5, 855-863, August 1993.-   Robert et al., Plant Molecular Biology 26, 1217-1222, 1994.-   Sato et al., The Plant Cell, Vol. 3, 867-876, September 1991.

1. A process for producing trangenic plants containing selectivelydestructable pistil- or stigma parts, wherein the plant possessesresistance to PCT as a result of phosphinothricir fixed precisely by thesequence. The protein sequence is defined by the DNA sequence, An ATGcodon, which is recognized in bacteria and plants, is used as thetranslation start codon; the Shine-Dalgarno sequence is underlined. Thisgene encodes the last step in the biosynthesis of PTT, i.e. thedeacetylation of inactive N-acetylphosphinothricin tripeptide to givethe active PTT, It is known that the specificity of many enzymes is notrestricted to one substrate. Thus, the phosphinothricinN-acetyltransferase which is encoded by the pat gene is actually used inPTT biosynthesis for acetylating desmethyl-PTC and, because of its lackof specificity, can be used for detoxifying PTC. By means ofoverexpressing the dea gene (using suitable promoters or by cloning ontohigh-copy vectors), an insufficiently specific N-acetyl-PTT deacetylasecan now be employed for activating N-acetylphosphinothricin, Other deagenes can be isolated from E. coli. Thus, it has been found that in E.coli, in contrast to other bacteria (e.g. rhizobias and streptomycetes),no activity can be detected in the so-called pat assay (dissertation ofInge Broer, University of Bielefeld Faculty of Biology, Expression desPhosphinthricin-N-Acetyltransferase-Gens aus Streptomycesviridochromogenes in Nicotiana tabacum (Expression of the Streptomycesviridochromogenes phosphinothricin N-acetyltransferase gene in Nicotianatabacum), pp. 42-43, 1989) after the pat gene has been cloned intosuitable expression vectors (Strauch et al., Gene, 63, 65-74, 1988;Wohlleben et al., Gene, 70, 25-37, 1988). In addition, when present inlow copy number in E. coli, the pat gene is unable to confer resistanceto PTT since the endogenous deacetylase nullifies the effect of thephosphinothricin N-acetyltransferase. Finally, this deacetylase activitycan be demonstrated directly by the efficient inhibition of GS activitywhich occurs after adding N-acetylphosinothricin. The deacetylaseconverts N-Ac-PTC into PTC, which then inhibits the GS in a knownmanner, as can be measured in a γ-glutamyltransferase assay (Bender etal., J. Bacteriol. 129, acetyltransferease activity, in addition, isgiven an N-Ac-PTC or N-Ac-PTT deacetylase gene under the control of apistil- or stigma specific promoter, and the pistil- or stigma arecaused to die by means of suitable, timely treatment with PTC or PTT. 2.The process according to claim 1, wherein the N-Ac-PTC or N-Ac-PTTdeacetylase gene is from Streptomyces viridochromogenes.
 3. The processaccording to claim 1, wherein the N-Ac-PTC or N-Ac-PTT deacetylase geneis from E. coli.